Graves'disease is one of the most common autoimmune diseases affecting humans, mainly women. In this disease, autoantib'odies generated by the immune system mimic the action of the pituitary hormone thyrotropin (TSH) thereby activating the TSH receptor on the thyroid gland and causing thyroid gland overactivity. Excess, unregulated thyroid hormone secretion leads to thyrotoxicosis with symptoms and signs affecting all organs in the body. Untreated, the disease can be fatal. Present therapy can treat but not cure Graves'disease. Moreover, there has been no significant advance in treatment in over 50 years. Recent evidence suggests that the molecular structure of the TSH receptor contributes to this autoimmune response. The goal of the present grant proposal is to enhance understanding of TSH receptor structure, particularly the extracellular region with which thyroid stimulating autoantibodies interact. The TSH receptor ectodomain comprises three components:- (i) an N-terminal domain (NTD) containing a cysteine cluster;(ii) a leucine-rich domain (LRD) comprising 9 leucine-rich repeats forming a slightly curved tubular structure. Each repeat has a beta-strand on its concave surface that forms part of the TSH binding region;(iii) a Hinge region bridging the LRD with the transmembrane region of the TSHR. The hinge is the least understood of these three TSHR ectodomain regions because of poor homology with other proteins of known structure. The TSHR hinge region is also unique among the glycoprotein hormone receptors in containing a -50 amino acid segment that is deleted during intramolecular cleavage into disulfide-linked A- and B-subunits. In contrast to the hinge region, the TSH receptor NTD and LRD structures are quite well understood because of the recent determination of the crystal structure of these FSH receptor components (lacking the hinge). However, the spatial inter-relationship between these three TSH receptor ectodomain components is unknown. The methodological approach to be used to investigate the TSH receptor will involve systematic mutagenesis of key amino acid residues chosen on the basis of present structural knowledge. The consequences of these mutations will be assessed structurally (using a large repertoire of monoclonal antibodies) and functionally (intracellular cAMP responses). The data obtained will provide insight into the pathophysiology of Graves'disease that will, hopefully, lead to improved forms of therapy in the future.